GPE analogs

ABSTRACT

The invention relates to GPE analogs, particularly GPE analogs capable of inducing an equivalent physiological effect to GPE within a patient. Such GPE analogs include peptides where the Gly of Gly-Pro-Glu is replaced by any of Ala, Ser, Thr, or Pro; where the Pro of Gly-Pro-Glu is replaced by any of Ala, Ser, Thr, or Gly; and where the Glu of Gly-Pro-Glu is replaced by any of Asn, Asp, or Gln. The GPE analogs of the invention have application in any method of therapy or prophylaxis in which GPE has application. These applications include the treatment of acute brain injury and neurodegenerative disease, including but not limited to injury or disease in the CNS. The GPE analogs will normally be administered as part of a pharmaceutical composition or preparation.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §371 to PCT/US01/41883,having an International Filing Date of Aug. 24, 2001, which claimedpriority to New Zealand Application Ser. No. 506,534, filed Aug. 24,2000, Each of the above applications is herein incorporated fully byreference.

This invention relates to GPE analogs.

BACKGROUND

GPE is a tri-peptide consisting of amino acids Gly-Pro-Glu. It and itsdi-peptide derivatives Gly-Pro and Pro-Glu were first disclosed by Saraet al in EP 0366638. Sara et al disclosed that GPE is effective as aneuromodulator (able to affect the electrical properties of neurons).

The applicants have also established that GPE has neuroprotectiveproperties and that it therefore has utility in the prevention orinhibition of neural cell death (WO 95/17204).

The nervous system contains neural cells and glial cells. Glial cells,including astrocytes, microglia, Schwann cells in the peripheral nervoussystem and oligodendrocytes in the central nervous system, often aidneural cells and neural activity by providing support and assistance toneural cells through means including anatomical configuration, metabolicactivity, and physiological function.

It is generally towards new molecules which mimic the functionality ofGPE that the present invention is directed. These molecules, which aretermed herein “GPE analogs” have application on an equivalent basis toGPE, including in treating and/or preventing neural damage followinginsult.

SUMMARY OF INVENTION

Accordingly, in a first aspect, the present invention provides amolecule which:

-   -   (i) has a neural binding-site profile which is at least        substantially equivalent to GPE; and    -   (ii) which has a neural bioactivity profile which is at least        substantially equivalent to that of GPE,        said molecule being other than GPE or its di-peptide derivatives        Gly-Pro and Pro-Glu.

Preferably, the molecule also has an ability to cross the blood brainbarrier in humans which is at least substantially equivalent to GPE.

In a further aspect, the present invention provides pharmaceuticalcompositions, particularly those adapted for peripheral or intrathecaladministration to a human patient, which comprise a molecule as definedabove.

In yet a further aspect, the invention provides a method of inducing aneuroprotective effect in a patient for therapy and/or prophylaxis whichcomprises the step of administering a molecules and/or a pharmaceuticalcomposition as defined above to said patient.

While the present invention is broadly as defined above, it will beappreciated by those persons skilled in the art that it also includesembodiments of which the following description provides examples. Inparticular, a better understanding of the present invention may beobtained through reference to the accompanying drawings in which:

FIG. 1 shows the binding site distribution of ³H-Me-GPE in therat-brain;

FIG. 2 shows the binding site distribution of ³H-Me-GPE in the humanbrain;

FIG. 3 is a graph showing accumulation of tritiated GPE in the brainover time; and

FIG. 4 shows the effect of GPE administered peripherally on neuronalsurvival after an hypoxic-ischemic injury.

DESCRIPTION OF THE INVENTION

As outlined above, the invention is broadly directed to molecules whichare GPE analogs. Such molecules will normally be structurally related toGPE and will in each case be capable of inducing an equivalentphysiological effect to GPE within a patient.

Fundamental to the invention is the recognition that it is possible tovary the structure, presentation and/or amino acid sequence of a proteinwhile retaining substantially equivalent functionality. For example, aprotein can be considered a functional equivalent of another protein fora specific function if the equivalent protein is immunologicallycross-reactive with the original protein. The equivalent can be, forexample, a fragment of a protein, a fusion of the protein with anotherprotein or carrier, or a fusion of a fragment with additional aminoacids. For example, it is possible to substitute amino acids in asequence with equivalent amino acids using conventional techniques.Groups of amino acids normally held to be equivalent are:

-   -   (a) Ala, Ser, Thr, Pro, Gly;    -   (b) Asn, Asp, Glu, Gln;    -   (c) His, Arg, Lys;    -   (d) Met, Leu, Ile, Val; and    -   (e) Phe, Tyr, Trp.

Thus, since GPE is the tripeptide Gly-Pro-Glu, GPE analogs includesubstitutions where the Gly of Gly-Pro-Glu is replaced by any of Ala,Ser, Thr, or Pro; where the Pro of Gly-Pro-Glu is replaced by any ofAla, Ser, Thr, or Gly; and where the Glu of Gly-Pro-Glu is replaced byany of Asn, Asp, or Gln. All of these equivalent molecules constituteGPE analogs of the invention.

The probability of one amino acid sequence being functionally equivalentto another can be measured by the computer algorithm BLASTP (Altschul etal 1990 J. Mol. Biol. 215: 403–410).

Further analogs in accordance with the invention include GPE amides andstearates. More particularly, specific analogs include the following:

-   -   GPE amide    -   GPE stearate    -   Gly-Pro-D-glutamate (GP-D-E)    -   Gly-Pro-Thr (GPT)    -   Gly-Glu-Pro (GEP)    -   Glu-Gly-Pro (EGP)    -   Glu-Pro-Gly (EPG),        all of which can be readily synthesized using standard        techniques.

Additional GPE analogs in accordance with the present invention will becharacterized by their being able to meet the following criteria:

-   -   having a neural binding-site profile which is at least        substantially equivalent to GPE    -   having a neural bioactivity profile which is at least        substantially equivalent to GPE    -   and desirably, although not essentially, having an ability to        cross the blood brain barrier (including in the absence of        neural insult) which is at least substantially equivalent to        GPE.

The neural binding-site profile for GPE has been determined as perExperiment 1 and is shown in FIGS. 1 and 2.

The ability of a candidate GPE analog to cross the blood brain barriercan be determined, and compared to passage rates for GPE, by followingthe procedures as set out in Experiment 2. In particular, theaccumulation of the candidate analog can be measured 60 minutesfollowing cardiac administration for ready comparison with GPE.

The neural bioactivity profile of a candidate GPE analog can bedetermined, and compared to that of GPE, by following the procedures setout in Experiment 3.

It is herein disclosed that GPE analogs can reduce neuronal cell lossdue to damage caused by an insult. An insult is damage, injury or stressthat may lead to death or dysfunction of nervous or glial cells ortissues. GPE analogs have application in any method of therapy orprophylaxis in which GPE has application. These include the treatment ofacute brain injury and neurodegenerative disease, including but notlimited to injury or disease in the CNS. For example, GPE analogs may beused in the treatment of multiple sclerosis. Such treatment may be acute(e.g., mainly directed to treating present symptoms) or may be chronic(e.g., mainly directed towards curing the disease, or towards preventingprogression of the disease and long-term amelioration of symptoms). Aprotective effect due to administration of a GPE analog prior to theinsult is termed a prophylactic effect, and a protective effect due toadministration of a GPE analog during or after the insult is termed aresuscitative effect. It will be appreciated that the protective effectsof GPE analogs, whether prophylactic or resuscitative, may be due toactions of GPE analogs on the neural cells themselves, on glial cells,or on cells of both types.

GPE analogs can be used in the manufacture of medicaments orpharmaceutical preparations for the treatment of medical conditions,including medical conditions resulting from neural injury or disease.Thus, GPE analogs can be administered as part of a medicament orpharmaceutical preparation. This can involve combining GPE analogs withany pharmaceutically appropriate carrier, adjuvant or excipient. Theselection of the carrier, adjuvant or excipient will of course usuallybe dependent upon the route of administration to be employed.

The administration route can vary widely. GPE analogs may beadministered in different ways, including subcutaneously,intraperitoneally, intravenously and intracerebroventricularly. GPEanalogs may be administered directly to a site of injury or of possibleinjury; for example, directly into the parenchyma of the brain or spinalcord. This can be achieved by any appropriate direct administrationroute. Examples of suitable methods include administration by lateralcerebroventricular injection or through a surgically inserted shunt intothe lateral cerebroventricle of the brain of the patient. Alternatively,GPE analogs may be administered to a location near to a site of injuryor of possible injury; or to a body cavity in contact or fluidcommunication with a site of injury or of possible injury (such as,e.g., a cerebral ventricle or to the cerebrospinal fluid bathing thespinal cord).

In addition, the ability of the GPE analogs to cross the blood brainbarrier allows them to be administered peripherally to a patient in needof treatment in the brain. The peripheral application may be the way ofchoice because then there is no direct interference with the centralnervous system.

Any peripheral route of administration known in the art can be employed.These can include parenteral routes for example injection into theperipheral circulation, subcutaneous, intraorbital, ophthalmic,intraspinal, intracisternal, topical, infusion (using eg. slow releasedevices or minipumps such as osmotic pumps or skin patches), implant,aerosol, inhalation, scarification, intraperitoneal, intracapsular,intramuscular, intranasal, oral, buccal, pulmonary, rectal or vaginal.The compositions can be formulated for parenteral administration tohumans or other mammals in therapeutically effective amounts (eg.amounts which eliminate or reduce the patient's pathological condition)to provide therapy for the neurological diseases described above.

Two of the most convenient administration routes include subcutaneousinjection (e.g., dissolved in 0.9% sodium chloride) and oraladminstration (e.g., in a capsule).

GPE analogs may be administered before or after an insult leading toneural injury or neural damage has occurred, or may be administeredconcurrently with such an insult. For example, GPE analogs may beadministered to a patient before a procedure or treatment which carriesrisk of neural damage in order to reduce or prevent any possible neuralinjury as result of the procedure or treatment. GPE analogs may also beadministered at any time up to and including about 100 hours after aninsult to protect neural or glial cells from injury or death. GPEanalogs may be particularly effective when administered during a timeperiod including the time between about 0.5 hours to about 8 hours afteran insult to protect neural cells from injury or death.

The calculation of the effective amount of GPE analogs to beadministered is within the skill of one of ordinary skill in the art,and will be routine to those persons skilled in the art. Needless tosay, the final amount to be administered will be dependent upon theroute of administration and upon the nature of the neurological disorderor condition that is to be treated. A suitable dose range may forexample be between about 0.01 mg to about 1 mg/100 g of body weight, ormore specifically about 0.06 μg to 0.6 mg of GPE analog per 100 g ofbody weight where the dose is administered centrally.

For inclusion in a medicament, GPE analogs can be directly synthesizedby conventional methods such as the stepwise solid phase synthesismethod of Merryfield et al., 1963 (J. Am. Chem. Soc. 15:2149–2154). Suchmethods of peptide synthesis are known in the art, and are described,e.g., in Fields and Colowick, 1997, Solid Phase Peptide Synthesis(Methods in Enzymology, vol. 289), Academic Press, San Diego, Calif.Alternatively synthesis can involve the use of commercially availablepeptide synthesizers such as the Applied Biosystems model 430A.

The starting materials and reagents used in preparing these compoundsare either available from commercial suppliers such as Aldrich ChemicalCompany (Milwaukee, Wis.), Bachem (Torrance, Calif.), Sigma (St.Louis,Mo.), or are prepared by methods well known to the person of ordinaryskill in the art following procedures described in such references asFieser and Fieser's Reagents for Organic Synthesis, vols 1–17, JohnWiley and Sons, New York, N.Y., 1991; Rodd's Chemistry of CarbonCompounds, vols. 1–5 and supplements, Elsevier Science Publishers, 1989;Organic Reactions, vols. 1–40, John Wiley and Sons, New York, N.Y.,1991; March J; Advanced Organic Chemistry, 4^(th) ed. John Wiley andSons, New York, N.Y., 1992; and Larock: Comprehensive OrganicTransformations, VCH Publishers, 1989. In most instances, amino acidsand their esters or amides, and protected amino acids, are widelycommercially available; and the preparation of modified amino acids andtheir amides or esters are extensively described in the chemical andbiochemical literature and thus well-known to persons of ordinary skillin the art.

The starting materials, intermediates, and compounds of this inventionmay be isolated and purified using conventional techniques, includingfiltration, distillation, crystallization, chromatography, and the like.They may be characterized using conventional methods, including physicalconstants and spectral data. Typically, the reactions described hereintake place at atmospheric pressure over a temperature range betweenabout 0° C. and 125° C.

Analogs of GPE, or modifications thereof, such as esters or amides, mayin general be prepared by methods such as are already well-known topersons of ordinary skill in the art of peptide and modified peptidesynthesis by following other methods well-known to those of ordinaryskill in the art of the synthesis of peptides and analogs.

Conveniently, synthetic production of the polypeptide of the inventionmay be according to the solid phase synthetic method described byMerrifield et al. Solid phase peptide synthesis. I. The synthesis of atetrapeptide: J. Amer. Chem. Soc. 85, 2149–2156, 1963. This technique iswell understood and is a common method for preparation of peptides. Thesolid phase method of synthesis involves the stepwise addition ofprotected amino acids to a growing peptide chain which is bound bycovalent bonds to a solid resin particle. By this procedure, reagentsand by-products are removed by filtration, thus eliminating thenecessity of purifying intermediates. The general concept of this methoddepends on attachment of the first amino acid of the chain to a solidpolymer by a covalent bond. Succeeding protected amino acids are added,one at a time (stepwise strategy), or in blocks (segment strategy),until the desired sequence is assembled. Finally, the protected peptideis removed from the solid resin support and the protecting groups arecleaved off.

The amino acids may be attached to any suitable polymer as a resin. Theresin must contain a functional group to which the first protected aminoacid can be firmly linked by a covalent bond. Various polymers aresuitable for this purpose, such as cellulose, polyvinyl alcohol,polymethylmethacrylate, and polystyrene. Suitable resins arecommercially available and well known to those of skill in the art.Appropriate protective groups usable in such synthesis includetert-butyloxycarbonyl (BOC), benzyl (Bzl), t-amyloxycarbonyl (Aoc),tosyl (Tos), o-bromo-phenylmethoxycarbonyl (BrZ), 2,6-dichlorobenzyl(BzlCl₂), and phenylmethoxycarbonyl (Z or CBZ). Additional protectivegroups are identified in Merrifield, cited above, as well as in McOmie JF W: Protective Groups in Organic Chemistry, Plenum Press, New York,1973.

The general procedure of preparation of the peptides of this inventioninvolves initially attaching the protected carboxyl-terminal amino acidto the resin. After attachment the resin is filtered, washed and theprotecting group (desirably BOC) on the α-amino group of thecarboxyl-terminal amino acid is removed. The removal of this protectinggroup must take place, of course, without breaking the bond between thatamino acid and the resin. The next amino, and if necessary, side chainprotected amino acid, is then coupled to the free α-amino group of theamino acid on the resin. This coupling takes place by the formation ofan amide bond between the free carboxyl group of the second amino acidand the amino group of the first amino acid attached to the resin. Thissequence of events is repeated with successive amino acids until allamino acids are attached to the resin. Finally, the protected peptide iscleaved from the resin and the protecting groups removed to reveal thedesired peptide. The cleavage techniques used to separate the peptidefrom the resin and to remove the protecting groups depend upon theselection of resin and protecting groups and are known to those familiarwith the art of peptide synthesis.

Alternative techniques for peptide synthesis are described in Bodanszkyet al, Peptide Synthesis, 2nd ed, John Wiley and Sons, New York, 1976.For example, the peptides of the invention may also be synthesized usingstandard solution peptide synthesis methodologies, involving eitherstepwise or block coupling of amino acids or peptide fragments usingchemical or enzymatic methods of amide bond formation. [See, e.g. H. D.Jakubke in The Peptides, Analysis, Synthesis, Biology, Acadermic Press,New York, 1987, p. 103–165; J. D. Glass, ibid., pp. 167–184; andEuropean Patent 0324659 A2, describing enzymatic peptide synthesismethods.] These solution synthesis methods are well known in the art.

A person of ordinary skill in the art will have no difficulty, takingaccount of that skill and the knowledge available, and of thisdisclosure, in developing one or more suitable synthetic methods forcompounds of this invention.

For example, analogs in which the glycine residue of GPE is replaced byan alternative amino acid may conveniently be prepared by thepreparation of a protected proline-glutamic acid di-peptide (such as thedibenzyl ester), and coupling that dipeptide with a protected glycineanalog, followed by deprotection. Analogs in which the glutamic acidresidue of GPE is replaced by an alternative amino acid or an amino acidamide or ester may conveniently be prepared by the preparation of aprotected glycine-L-proline di-peptide (such as BOC-glycyl-L-proline),and coupling that dipeptide with a protected glutamic acid and esters orglutamine. Where modifications are to be made to two or more aminoacids, the coupling techniques will still be the same, with just morethan one different amino acid or analog being used in the synthesis. Thechoice of appropriate protecting groups for the method chosen(solid-phase or solution-phase), and of appropriate substrates ifsolid-phase synthesis is used, will be within the skill of a person ofordinary skill in the art.

Conjugation and modification of the resulting peptides may beaccomplished by standard techniques known in the art. For example,esterification may be used to provide GPE-stearate, and amidation may beused to provide GPE-amide. GPE analogs may be made by conjugatinganother molecule to GPE. For example, it is known in the art toconjugate polyethylene glycol (PEG) to peptides; such modified peptidesare termed PEGylated peptides. PEGylated GPE peptides comprise GPEanalogs of the invention, and may be provided using, e.g., the conjugatetechnology described in WO 95/32003 published Nov. 30, 1995. Variousweights of PEG may be used to provide a variety of PEGylated peptideanalogs. PEGylated peptides often remain available within a patient forlonger times than nonPEGylated peptides.

As a general proposition, the total pharmaceutically effective amount ofGPE analog administered parenterally per dose will be in a range thatcan be measured by a dose response curve. The preferred range will bebetween about 0.01 mg to about 1 mg per 100 g body weight, specificallybetween about 0.06 mg/100 g body weight to about 0.6 mg/100 g bodyweight. For example, GPE analogs in the blood can be measured in bodyfluids of the mammal to be treated to determine dosing. Alternatively,one can administer increasing amounts of the GPE analog to the patientand check the serum levels of the patient for the GPE analog. The amountof GPE analog to be employed can be calculated on a molar basis based onthese serum levels of GPE analog.

Specifically, one method for determining appropriate dosing of theanalog entails measuring GPE analog levels in a biological fluid such asa body or blood fluid. Measuring such levels can be done by any means,including RIA and ELISA. After measuring GPE analog levels, the fluid iscontacted with the compound using single or multiple doses. After thiscontacting step, the GPE analog levels are re-measured in the fluid. Ifthe fluid GPE analog levels have fallen by an amount sufficient toproduce the desired efficacy for which the molecule is to beadministered, then the dose of the molecule can be adjusted to producemaximal efficacy. This method can be carried out in vitro or in vivo.Preferably, this method is carried out in vivo, i.e. after the fluid isextracted from a mammal and the GPE analog levels measured, the analogherein is administered to the mammal using single or multiple doses(that is, the contacting step is achieved by administration to a mammal)and then the GPE analog levels are remeasured from fluid extracted fromthe mammal.

GPE analogs may also be suitably administered by a sustained-releasesystem. Suitable examples of sustained-release compositions includesemi-permeable polymer matrices in the form of shaped articles, e.g.,films, or microcapsules. Sustained-release matrices include polylactides(U.S. Pat. No. 3,773,919; EP 58,481), poly(2-hydroxyethyl methacrylate)(Langer et al., 1981), ethylene vinyl acetate (Langer et al., supra), orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988). Sustained-releasecompositions also include a liposomally entrapped compound. Liposomescontaining GPE analogs are prepared by methods known per se: DE3,218,121; Hwang et al., 1980; EP 52,322; EP 36,676; EP 88,046; EP143,949; EP 142,641; Japanese Pat. Appln. 83-118008; U.S. Pat. Nos.4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes areof the small (from or about 200 to 800 Angstroms) unilamellar type inwhich the lipid content is greater than about 30 mol percentcholesterol, the selected proportion being adjusted for the mostefficacious therapy. All documents referred to herein, both supra andinfra, are hereby incorporated by reference in their entirety.

For parenteral administration, doses may be between about 0.01 to about1 mg of GPE analog per 100 g of body weight, more specifically about0.06 μg to 0.6 mg of GPE analog per 10 g body weight. In one embodiment,the analog is formulated generally by mixing each at the desired degreeof purity, in a unit dosage injectable form (solution, suspension, oremulsion), with a pharmaceutically, or parenterally, acceptable carrier,i.e., one that is non-toxic to recipients at the dosages andconcentrations employed and is compatible with other ingredients of theformulation. For example, the formulation preferably does not includeoxidizing agents and other compounds that are known to be deleterious topolypeptides.

Generally, the formulations are prepared by contacting the compounduniformly and intimately with liquid carriers or finely divided solidcarriers or both. Then, if necessary, the product is shaped into thedesired formulation. Preferably the carrier is a parenteral carrier,more preferably a solution that is isotonic with the blood of therecipient. Examples of such carrier vehicles include water, saline,Ringer's solution, a buffered solution, and dextrose solution.Non-aqueous vehicles such as fixed oils and ethyl oleate are also usefulherein.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; glycine; amino acids such as glutamic acid,aspartic acid, histidine, or arginine; monosaccharides, disaccharides,and other carbohydrates including cellulose or its derivatives, glucose,mannose, trehalose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; counter-ions such as sodium;non-ionic surfactants such as polysorbates, poloxamers, or polyethyleneglycol (PEG); and/or neutral salts, e.g., NaCl, KCl, MgCl₂, CaCl₂, etc.

The GPE analog is typically formulated in such vehicles at a pH of fromor about 4.5 to 8. It will be understood that use of certain of theforegoing excipients, carriers, or stabilizers will result in theformation of salts of the compound. The final preparation may be astable liquid or lyophilized solid.

Typical adjuvants which may be incorporated into tablets, capsules, andthe like are a binder such as acacia, corn starch, or gelatin; anexcipient such as microcrystalline cellulose; a disintegrating agentlike corn starch or alginic acid; a lubricant such as magnesiumstearate; a sweetening agent such as sucrose or lactose; a flavoringagent such as peppermint, wintergreen, or cherry. When the dosage formis a capsule, in addition to the above materials, it may also contain aliquid carrier such as a fatty oil. Other materials of various types maybe used as coatings or as modifiers of the physical form of the dosageunit. A syrup or elixir may contain the active compound, a sweetenersuch as sucrose, preservatives like propyl paraben, a coloring agent,and a flavoring agent such as cherry. Sterile compositions for injectioncan be formulated according to conventional pharmaceutical practice. Forexample, dissolution or suspension of the active compound in a vehiclesuch as water or naturally occurring vegetable oil like sesame, peanut,or cottonseed oil or a synthetic fatty vehicle like ethyl oleate or thelike may be desired. Buffers, preservatives, antioxidants, and the likecan be incorporated according to accepted pharmaceutical practice.

Typically, the GPE analog to be used for therapeutic administration mustbe sterile. Sterility is readily accomplished by filtration throughsterile filtration membranes (e.g., 0.2 micron membranes). Therapeuticcompositions generally are placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle.

The GPE analog ordinarily will be stored in unit or multi-dosecontainers, for example, sealed ampules or vials, as an aqueous solutionor as a lyophilized formulation for reconstitution. As an example of alyophilized formulation, 10 mL vials are filled with 5 ml ofsterile-filtered 0.001% (w/v) aqueous solution of compound, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized compound using bacteriostaticWater-for-Injection.

EXPERIMENTAL Experiment 1

Materials and Methods

Synthesis of tritium-labelled GPE (³H-Me-GPE)

A Schiff base formed between formaldehyde and the N-terminal glycineamino group of GPE tripeptide at 0–4° C., pH 8.5 was reduced with highspecific activity tritiated sodium borohydride (100 mCi, 50–70 Ci/mM,Amersham, Bucks, UK) resulting in the addition of a stabletritium-bearing methyl group on the free amine of the glycine asdescribed previously (Means, G. E. and Feeney, R. E. in Chemicalmodification of proteins, Vol 1, 216–217, Holden-day, Inc, SanFrancisco; Guan et al., 1996, NeuroReport, Vol. 7).

After tritiation the reaction mixture was diluted in ammonium acetatebuffer (0.05M, pH 5.5 with 20% v/v methanol, 10 ml) and purified usingAccell CM- and QMA+Sep-Paks (Waters, Milford, Mass., USA) in series.After washing with the ammonium acetate buffer to remove reagents andbyproducts (tritiated water and tritiated methanol), the CM- andQMA+cartridges were uncoupled and the ³H-Me-GPE was eluted from the QMAcartridge with ammonium bicarbonate (0.3M, 6 ml). The eluted materialwas lyophilized twice to remove the bulk of the ammonium bicarbonate,dissolved in ethanol-water (1:19, 20 μl ) and aliquots stored at −70° C.

Animal Preparation

The following experimental protocol followed guidelines approved by theUniversity of Auckland Animal Ethics Committee. Adult male Wistar ratswere killed by decapitation and tissues immediately removed and storedat −80° C. Coronal sections (16 μm) were cut on a cryostat and mountedon gelatin-coated slides. Normal adult human postmortem brain (n=4)tissue was cut and mounted in a similar manner.

The optimal protocol for ³H-Me-GPE binding was: a 10 minutepreincubation in buffer (50 nM Tris-HCl, pH 7.4) with proteaseinhibitors (20 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 mMN-ethylmaleimide and 5 mM benzamidine), followed by a 90 minuteincubation at 24° C. in the same buffer including 50 nM ³H-Me-GPE,followed by 2×1 minute washes in ice-cold distilled water and dried in acold air flow. Non-specific binding was defined by coincubation with theunlabeled ligand (10 μM) and was typically 25–30%.

Results

The results are shown in FIGS. 1 and 2.

The observed distribution of ³H-Me-GPE appears to be unique incomparison to that of related ligands (Monaghan, D. T. and Cotman, C.W., J Neuroscience, Vol. 5, 1985; Bohannon et al., Brain Research, Vol.444, 1988; Wenzel et al., NeuroReport, Vol. 7, 1995). There was intensebinding in the CA1-2 region of the hippocampus and lesser binding in thepyriform cortex, amygdala, choroid plexus and large blood vessels (FIG.1). In comparison to the CA1-2 region of the hippocampus there was lessbinding in the molecular layer of the dentate gyrus, where glutamate(NMDA) receptors are present at high density (Monaghan, D. T. andCotman, C. W., J Neuroscience, Vol. 5, 1985). Binding in the CA1-2region was blocked by an excess (10 μM) of N-methyl-GPE but not byglutamate (1 mM) or by MK-801, NBQX, glycine, nicotine or adenosine (100μM). The highest binding in the rat brain was seen in the strataradiatum and oriens of CA1. Apart from a trace of binding in the lungand major blood vessels, ³H-Me-GPE-binding activity was not detected inany other tissue.

To determine whether there was a similar distribution of binding sitesin the human brain autoradiography of ³H-Me-GPE was performed onsections spanning the hippocampus and entorhinal cortex. The highestbinding was observed in the molecular layer of the dentate gyrus and theCA1-2 region of the hippocampus (FIG. 2).

Experiment 2

Materials and Methods

Tritiated GPE (Gly-[³H]Pro-Glu, 1.6 ml, specific activity 50–60 Ci/mmol)obtained from Sibtech, Inc., USA was used in these experiments.

Animal Preparation

The following experimental protocol followed guidelines approved by theUniversity of Auckland Animal Ethics Committee.

Cardiac Administration

Adult Wistar rats (300 g) were prepared under halothane/O₂ anaesthesia.The rats were randomly assigned to one of four groups: tritiated GPE,saline+0.1% BSA, tritiated GPE+non-tritiated GPE or tritiated proline.Rats in the tritiated GPE, tritiated proline and control groups weresacrificed at 30 min, 1 hr or 6 hrs after administration. Ratsco-administered with both tritiated and non- tritiated GPE weresacrificed at 10 minutes after administration. For all groups, tissuewas homogenized in Soluene® (a tissue solubilizer; Packard BioscienceCompany, Meriden Conn.) and counted in a β counter.

V and IP Administration

Rats were injected with 10 million counts of tritiated GPE in 500 μltotal (made up with 0.1% BSA in saline). Animals were sacrificed at 30minutes, 1, 3 or 6 hrs. Tissue was homogenized in Soluene® and countedin a β counter.

ICV Administration

Adult Wistar rats (300 g) were prepared under halothane/O₂ anaesthesia.A guide cannula was placed on the dura 7.5 mm anterior from stereotaxiczero and 1.5 mm from midline on the right. One million counts of GPE and600 μl of non-tritiated GPE were injected in 8 μl total volume (made upwith 0.1% BSA in saline). Rats were sacrificed 30 minutes afteradministration. Tissue was homogenized in Soluene® and counted in a βcounter.

Tritiated GPE, tritiated GPE+non-tritiated GPE, tritiated proline(Amersham) or tritiated proline+non-tritiated proline in vehicle (0.1Mcitrate buffer [pH6], diluted 10 times in 0.1% bovine serum albumin in0.1M phosphate buffered saline [PBS] [pH7.3]) were then given bydifferent routes of administration to injured and non-injured ratsaccording to the following study design:

1) cardiac puncture: tritiated GPE  10 million counts (20 μl in 300 μl)tritiated and non-tritiated GPE  10 million counts + 600 μg tritiatedproline  10 million counts Saline + 0.1% BSA 200 μl 2) intravenously(iv): tritiated GPE  10 million counts Saline + 0.1% BSA 200 μl 3)intraperitoneally (ip): tritiated GPE  10 million counts Saline + 0.1%BSA 200 μl 4) intracerebroventricularly (icv): tritiated GPE  1 millioncounts tritiated and non-tritiated GPE  1 million counts + 600 μg

The following describes the various methods of the routes ofadministration for the administration of the following solutions:tritiated GPE, tritiated GPE+non-tritiated GPE, tritiated proline, ortritiated proline+non-tritiated proline.

Cardiac administration was performed using a syringe. The syringe needlewas placed into the left ventricle and one of the above four solutionswas given.

IV administration was carried out by injecting one of the four solutionsinto the tail vein.

IP administration was performed by injecting one of the four solutionsinto the peritoneal cavity.

ICV administration was carried out according to the following procedure.The rats were lightly anaesthetized again using 0.15 ml Saffan™(Pitman-Moore Ltd, NZ). The infusion of one of the four solutions wasmade into the right lateral ventricle guided by a metal cap fitted overthe rat head using a modified technique originally described byJirikowski (J Neuroscience Methods, 42: 115–118, 1992), in order toensure correct placement of the infusion needle. One of the foursolutions was administered in a single dose.

The rats were sacrificed using pentobarbital 10, 60 or 360 minutes afteradministration of either tritiated GPE or tritiated proline. Thedistribution of administered tritiated GPE or proline was ascertained.Counts/minute/mg were made in the following tissues: kidney, blood,adrenal glands, liver, lung, testicle, heart, muscle, spinal cord andbrain. HPLC was used in order to check that the particles counted wereeither tritiated GPE or tritiated proline.

Results

The amount of tritiated GPE increases over time in the brain relative tothe blood (FIG. 3). With cardiac administration 60 minutes was thetime-point at which there was the most tritiated GPE in the brain.

When tritiated and non-tritiated GPE were administered both together viacardiac administration the only displacement of GPE occurred in thebrain. This result was supported by ICV administration.

Furthermore, using ICV administration GPE was found to be selectivelytaken up on the injured side of the brain in rats with anhypoxic-ischemic brain injury.

With ip administration the amount of GPE in the brain compared to thecorresponding time-points after iv and cardiac injection was increased.

These results therefore show the ability of GPE to pass through theblood-brain-barrier, including in the absence of neural insult.

Experiment 3

Materials and Methods

This experiment involved treating rats with a control vehicle or GPEadministered peripherally 2 hours after a focal CNS injury. The rats hadan hypoxic-ischemic injury to one cerebral hemisphere induced in astandard manner (ligation of the carotid artery). The degree and lengthof hypoxia, the ambient temperature and humidity were defmed tostandardize the degree of damage. The neuronal death is restricted tothe side of the carotid ligation and is primarily in the hippocampus,dentate gyrus, striatum and lateral cortex of the ligated hemisphere.There is no neuronal loss in the contralateral hemisphere.

Specifically forty-nine 50–60 day old adult Wistar rats (280–310 g) wereprepared under halothane/O₂ anaesthesia. The right side carotid arterywas ligated. The rats were allowed to recover for 1 hour and were thenplaced in an incubator with humidity 90±5% and temperature 31±0.5° C.for 1 hour before hypoxia. Oxygen concentration was reduced andmaintained at 6±0.2% 0₂ for 10 minutes. The rats were kept in theincubator for 2 hours after hypoxia and then treated either with Smg GPEadministered intraperitoneally or vehicle alone (saline+0.1% BSA). Therats were sacrificed using pentobarbital 7 days after hypoxic-ischemicinjury.

The rats were transcardially perfised with 0.9% saline followed by 4%paraformaldehyde, and the brains were removed and embedded in paraffin.Symmetric serial coronal sections (4 μm) were cut and stained withthionin/acid-fuchsin for live/dead neurons (Sirimanne et al., 1994Journal of Neuroscience, 55: 7–14). The histological outcome of neuronalsurvival was examined with light microscopy (Leica) in the hippocampusin the injured hald of the brain according to a reference of rat brainanatomy (Paxinos, and Watson (1982) The rat brain in stereotaxiccoordinates, 2^(nd) Edition, Academic Press, New York, USA). Only cellswith a morphology like live neurons were counted, while dead neurons orcells with morphology like glia were not included. One coronal sectionwas used for each brain.

A coronal section (A-P 4.5 mm) was used for analysis of the hippocampus.All surviving neurons in the hippocampal CA1/2 region of the injuredhemisphere were counted. Data were analysed with paired t-test andpresented as mean±sem (standard error of the mean).

Results

The results are shown in FIG. 4.

GPE administered peripherally increases neuronal survival in thehippocampus after an hypoxic-ischemic injury.

These results establish a neural bioactivity profile for GPE againstwhich a candidate analog can be compared.

UTILITY

Thus, in accordance with the present invention there are provided GPEanalogs. These analogs have application in any method of therapy orprophylaxis in which GPE has application. These include the treatment ofacute brain injury and neurodegenerative disease, including but notlimited to injury or disease in the CNS.

In addition, the ability of the GPE analogs to cross the blood brainbarrier allows them to be administered peripherally to a patient in needof treatment in the brain.

As with GPE, the analogs will normally be administered as part of apharmaceutical composition or preparation.

Those persons skilled in the art will appreciate that the presentinvention is described above by way of example only and is not intendedto be limited to the specific experimental details given.

1. An analog of the tripeptide, Gly-Pro-Glu (GPE), wherein said Gly isreplaced by Ala thereby fanning L-Ala-PE.
 2. A method of treating amedical condition resulting from neural injury or disease, comprisingadministering an effective amount of L-Ala-PE.
 3. The method of claim 2,wherein said medical condition is associated with hypoxic-ischemicinjury.
 4. The method of claim 2, wherein said disease is multiplesclerosis.
 5. The method of claim 2, wherein said L-Ala-PE isadministered at any time up to and including about 100 hours after saidneural injury or disease.
 6. The method of claim 2, wherein saidL-Ala-PE is administered prophylactically.
 7. The method of claim 2,wherein said medical condition results from a procedure likely toproduce acute brain injury.
 8. The method of claim 7, wherein saidL-Ala-PE is administered prior to said procedure.
 9. A pharmaceuticalcomposition comprising L-Ala-Pro-Glu (L-Ala-PE) and a pharmaceuticallyacceptable carrier.
 10. The pharmaceutical composition of claim 9, and apharmaceutically acceptable excipient.
 11. A method of protectingneuronal cells in a patient from damage likely to result in neuralinjury or disease, comprising administering to said patient atherapeutic amount of a GPE analog of claim
 1. 12. The method of claim11, wherein said cells are neural cells.
 13. The method of claim 11,wherein said damage is associated with an elective procedure.
 14. Themethod of claim 11, wherein said L-Ala PE is included with apharmaceutically acceptable excipient as a pharmaceutical composition.15. A pharmaceutical composition comprising the analog of claim 1, andat least one pharmaceutically acceptable excipient selected from thegroup consisting of water, Ringer's solution, dextrose, phosphatebuffer, citrate buffer, succinate buffer, acetic acid buffer, anantioxidant, peptides having a less than about 10 amino acid residues,polyarginine, tripeptides, serum albumin, gelatin, immunoglobulin,polyvinylpyrrolidone, glycine, amino acid, monosaccharide, disaccharide,cellulose, glucose, mannose, trehalose, dextrins, chelating agents,sugar alcohols, counter ions, non-ionic surfactants and neutral salts.16. The pharmaceutical composition of claim 9, wherein saidpharmaceutical composition is adapted for peripheral administration. 17.The pharmaceutical composition of claim 9, wherein said pharmaceuticalcomposition is adapted for intrathecal administration.
 18. The method ofclaim 11, wherein the route of administration is by implant, aerosol,inhalation, scarification, intraperitoneal, subcutaneous, intracapsular,intramuscular, intranasal, oral, buccal, pulmonary, rectal, vaginal orintravenous.
 19. The method of claim 11, wherein said GPE analog isadministered in a dose range of: from about 0.01 mg/100 g body weight;to about 1 mg/100 g body weight.
 20. The method of claim 11, whereinsaid damage is associated with an elective surgical procedure.
 21. Thepharmaceutical composition of claim 9, in a capsule.